The role of protein phosphorylation in the regulation of sperm flagellar motility by cAMP and calcium will be studied. A heat-stable NP-40-soluble 56,000 Mr protein whose cAMP-dependent phosphorylation is required for flagellar motility has been identified. In addition, calcium-calmodulin appears to regulate flagellar motility at the level of the sperm membranes where it controls intracellular calcium levels. The specific aims for the next budget period of the project are: (1) Purify and raise antibodies to the 56,000 Mr phosphoprotein and the membrane calmodulin binding proteins; (2) Determine the site and mechanism of action of the 56,000 Mr phosphoprotein in the regulation of flagellar motility and how alterations in the phosphorylation state of the protein affects its function; (3) Investigate the significance of this protein with respect to time of synthesis and phosphorylation during spermatogenesis in relation to the acquisition of motility during sperm maturation; (4) Identify and determine the role of the sperm membrane calmodulin binding proteins in the regulation of flagellar motility and whether the phosphorylation of these proteins affects their calmodulin dependency and function; (5) Explore the possible role of other cAMP-dependent and -independent phosphoproteins and calmodulin binding proteins in the cAMP and calcium-dependent regulation of flagellar motility and relate these proteins to the suggested calmodulin-independent inhibition of motility by calcium. The major experimental approach to pursue these specific aims comprises the use of detergent-permeabilized sperm reactivated with (alpha-32P)ATP. Specific parameters of flagellar motility will be modified by the addition of specific protein and antibody probes and be quantitated by video image analysis. Protein phosphorylation will be measured under identical experimental conditions by high resolution 2-dimensional polyacrylamide gel electrophoresis, autoradiography and digital image processing. The results obtained should yield a greater understanding of the basic regulatory mechanisms involved in the control of axonemal motility and identify possible abberations underlying disease states typified by altered axonemal motility.